Preparation Of B-Lactoglobulin And Structure Study Of Its Selenious Acid Compound

This study was aimed to find a new kind of organic selenium compound of β-1-actoglobulin and selenium dioxide in the vacuum and low temperature conditions. T-he structure changes of selenious-β-lactoglobulin were revealed by fourier transform infrared spectroscopy (FT-IR),circular dichroism(CD), X-ray diffraction(X-ray),nuclear magnetic resonance spectrum(NMR),MALDI-TOF-MS,two-dimensional electrophoresi-s(2D-PAGE),differential scanning calorimetry(DSC).Fractionation of β-lactoglobulin from milk was explored by ammonium sulfate fractionation,sephadexG-50 column chromatography and optimized membrane separation procedure.The result showed that 40%-70% ammonium sulfate could enrich most P-lactoglobulin. It was the optimum condition of SephadexG-50 column chromatography that the sample concentration was 20mg/mL, sample volume was 2 mL and flow rate was 4s per drops.The optimum conditions of ultrafiltration were pressure 0.25Mpa, time 50min.The purity of β-lactoglobulin was determined by the Kjeldahl method and 2D-PAGE and it could reach 92.5%.Blended P-lactoglobulin with selenium dioxide,adjusted pH to l,reacted in-0.1Mpa for 5 hours.Finally got selenious-β-lactoglobulin, its selenium content was 15.17mg/g. Selenious-β-lactoglobulin was added strong bands at 878cm-1 and 1043cm-1, which belonged to Se=O and Se-O, compared to β-lactoglobulin by FT-IR.Finally, the structure changes of selenious-P-lactoglobulin were studied. CD spectra results showed that, compared with the β-lactoglobulin, the secondary structure of selenious-P-lactoglobulin was changed significantly and it included a-helical ratio increased 9%,P-sheet reduced of 14.2%,β-turns decreased 6% and random coil increased 11.2%. The consequence was also demonstrated by X-ray. The results of DSC showed that the thermal stability was reduced. NMR analysis results also showed that the tertiary structure was changed.2D-PAGE displayed that selenious-β-lactoglobulin contained two proteins, which the molecules quantity of one is two times of another. One-dimensional peptide fingerprint of the two proteins were highly consistent by M ALDI-TOF-MS.It could be concluded that some selenium dioxide only connected with one P-lactoglobulin, the other could connect with two β-lactoglobulins.Above all, selenious-β-lactoglobulin could be generated by selenium dioxide and β-lactoglobulins under the conditions of vacuum and low temperature. Both secondary structure and tertiary structure was changed in P-lactoglobulin when it reacted with selenium dioxide. This study would lay the foundation for the functional study of selenio us-β-lacto globulin.