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| Author(s): |
Marcelo Anselmo Oseas da Silva
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Química |
| Defense date: | 2008-02-25 |
| Examining board members: |
Marco Aurelio Zezzi Arruda;
Maria Fernanda Georgina Gine Rosias;
Lauro Tatsuo Kubota
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| Advisor: | Marco Aurelio Zezzi Arruda |
| Abstract | |
In this work the partition behavior of albumin was studied, which is found at high concentrations in blood plasma, which interferes in many analytical techniques determinations. The present study evaluated different aqueous two-phase systems, exploiting a phenomenon called of cloud point. These systems employ surfactants under specific experimental conditions, enabling formation of two immiscible phases: one rich and another poor in surfactant. Due to the hydrophilic characteristcs of albumin, as well as its aggregate dimensions, its extration to the surfactant rich phase presented partition coefficients lower than 0.66, representing and extraction efficiency of ca. 40%. Protein extraction was feasible by applying a mixture comprised of a nonionic surfactant (Triton® X-114), which allowed the phase separation at biocompatible temperatures, and an ionic one (sodium dodecylsufate - SDS), wich promotes eletrostatic interactions between aggregates and protein molecules, since the extraction procedure is carried out at pH 5.0. Circular dichroism analysis complemented the study and it showed that a system based on a Triton® X-114 and SDS mixture causes partial protein denaturation, but its application for a real sample is feasible. The efficiency of this was evaluated for albumin removal from blood plasma. A partition coefficient of 1.1 was obtained, indicating that ca. 51% of proteins were contained in the surfactant rich phase. Albumin depleted samples were submitted to gel eletrophoresis and the surfactant poor phase presented a more detailed gel electrophoresis profile, when compared with a crude sample. The surfactant rich phase reveled that albumin is the predominant protein present, but it is possible to find other highly concentrated plasmatic proteins including immunoglobulin G and transferrin. Finally, the method presented similar performance when compared with commercially available systems for albumin removal, such as the Millipore® system, wich was also evaluated in this work (AU) | |