Extração e fracionamento de proteinas de plasma sanguineo baseada no procedimento do ponto nuvem / Extration and fractionation of blood plasma proteins based on cloud point procedure

AUTOR(ES)

Aline Soriano Lopes

DATA DE PUBLICAÇÃO

2005

RESUMO

The goal of the present work is to fractionate the human blood plasma proteins using micelle-mediated phase separation with Triton X -114 as surfactant and NaCI as electrolyte. Some parameters that affect the phase separation are evaluated, such as Triton X-114 concentration (2 - 15% w/v), sample volume (50 - 2000 mL), NaCI concentration (6 - 12% w/v), contact time between proteins and micelles aggregation (0 - 30 min) and pH (6.4 - 7.4). The total proteins are quantified in both surfactant-rich and -poor phases by using the Bradford method. The surfactant present in the rich phase is removed with ice-cold acetone before the protein determination, in which the proteins are precipitated and the surfactant is removed to the organic phase. The separation is carried out at room temperature (25 °C) and the optimized parameters were 5% (w/v) Triton X-114, 100 mL of sample volume, 10% (w/v) NaCI, contact time almost instantaneous and pH as 6.8. Protein characterization is made through 1D electrophoresis. The software Gel-Pro Analyzer 3.1 is used for analyzing the bands as well as the protein data bank Expasy is used for identifies the possible protein fractions. Two bands are observed in the surfactant-rich phase with measurable concentrations. One of them may be attributed to albumin, which is present at highest concentration in blood plasma, and the other one may be attributed to apolipoprotein D or immunoglobulin light chain. Bovine serum albumin standard is used as reference for emphasizing possible changes on the secondary structure of proteins by circular dichroism. Changes were observed in the albumin conformation when the protein is submitted to phase separation with Triton X-114.

ASSUNTO(S)

proteina protein fracionamento fractionation ponto nuvem cloud point plasma plasma

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