Reverse Micelle System Of High-speed Countercurrent Chromatography Separation Of Proteins And Peptides

In this paper, high-speed counter-current chromatography (HSCCC) was developed for the separation and purification of protein mixtures and almond antihypertensive peptides. Since most protein and peptide molecules are not soluble in organic solvents, special solvent systems are required in CCC separations. There are two types of solvent systems meeting the requirements:two-phase aqueous system, reverse micellar solution. Two-phase aqueous system has been widely employed in CCC for protein separations. But employing micellar solution in protein separation by HSCCC has not been reported yet. This paper was exploratory research about the separation of protein mixture with gradient or isocratic elution, and the separation of almond antihypertensive peptides. Meanwhile, the puirity of protein was identified by means of high performance liquid chromatography.There are five parts of this paper:1. ReviewFirstly, high-speed counter-current chromatography was introduced from four aspects, which included principles, advantages, how to select two-phase solvent system and the research status; Then, did a summary of the concept, type, extractional principle and its affect ingredients of reverse micelles. Also, the application of separating protein and peptide using reversed micelles extraction was introduced; Then, did a overview of struture and function of several common proteins and separation methods of protein mixtures; Finally, did a breif overview of biological function and purification methods of bioactive peptides.2. Separation of protein mixtures by HSCCC with isocratic elutionWe used 0.02 mol/L AOT/Span80-n-hexane (1:1000 v/v) reversed micelle solution as stationary phase. When separated under isocratic elution, not only BSA and collagen, but also bovine hemoglobin and BSA were eluted together. The former protein mixture was not separated under 0.05 M Tris-HCl, pH 6.97 with 0.1 M KCl and 0.05 M Na2HPO4-NaH2PO4, pH 6.00 with 0.1 M KCl. The latter protein mixture was not separated under 0.05 M Tris-HCl, pH 9.00 with 0.1 M KC1.3. Protein separation by HSCCC using reverse micelle solvent systemsBy comparing the stationary phase retention ratio of twelve kinds of reverse micelle solvent systems, we choosed solvent systems composed of AOT+Span80-n hexane-water for our purification in HSCCC. The stationary phase was prepared by dissolving AOT and Span80 in n-hexane. The mobile phases were buffer solutions. Considering the loss of stationary phase, we determined to add 0.5 ml Span80 in 500 ml n-hexane; according to single factor tests, the results were as follow:revolution speed,650 rpm; flow-rate,1.5 ml/min; the first mobile phase,0.05 M Tris-HCl, pH 7.35 with 0.1 M KCl and the second mobile phase,0.05 M Na2HPO4-NaOH, pH 11.95 with 0.8 M KCl. After identifying the two fractions by HPLC, the results were that:the first fraction was BSA with purity over 94.3%; the second fraction was lysozyme with purity over 96.6%.4. Antihypertensive peptides separation by HSCCC using reverse micelle solvent systems from almondThe fresh almonds were removed the peel, dried in air, ground to powders and extracted with base solution after defateed with n-hexane. The almond proteins were hydrolyzed by Alcalase, and fractionated through UF membranes with a range of molecular weight cutoffs (MWCO). Under the 1 kDa fraction showed the highest ACE inhibitory activity, and was preliminarily separated by HPLC. The result indicated that almond peptides under 1 kDa were complex. Finally, we separated the above almond peptides by HSCCC with AOT+Span80-n hexane-water as micellar solvent systems. Though separation was truly achieved using pH gradient by the presence of the micellar phase in the stationary phase, test had poor reproducibility.5. research summaryIn this part, it summaried the results of tests, and predicted its future development.