Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

Abstract

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ionexchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented ³99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60 o C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37 o C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

The homotrimeric structure of HtrA2 is indispensable for executing its serine protease activity

“…The protein bands were detected by staining of the gel with Coomassie Brilliant Blue dye. The electrophoretic mobility of proteins was analyzed by comparison with proteins with known molecular masses (Tanaka et al, 2001). …”

Ammonium sulfate precipitation combined with liquid chromatography is sufficient for purification of bovine serum albumin that is suitable for most routine laboratory applications

“…For its use as a stabilizer and blocking agent, the main drawback is contamination with DNase and IgGs. More stringent purification steps are required to make BSA preparations suitable for use in blood transfusion and other clinical applications [4,12]. Even though BSA is in high abundance in cow blood, commercial purification of molecular grade BSA involves several cumbersome and time-consuming steps [14].…”

“…Bovine serum albumin (BSA) is widely used in various biochemical and immunological applications due to its high abundance in bovine plasma, structural stability, and its noninvasive stabilizing interaction with most proteins [1][2][3][4][5]. Albumin is used as a carrier for several molecules in drug delivery systems, a stabilizing protein to dilute antibodies as well as a blocking agent in various immunoassays and detection protocols [1][2][3][4][5].…”

“…Albumin is used as a carrier for several molecules in drug delivery systems, a stabilizing protein to dilute antibodies as well as a blocking agent in various immunoassays and detection protocols [1][2][3][4][5]. Most current approaches to purification of BSA combine the original or modified version of the Cohn method [6,7] with several additional steps such as ion exchange chromatography [8][9][10][11][12][13][14].…”

Identification of potential targets for an anticoagulant pectin

“…This limitation motivated us in establishing a simple and non-expensive protocol for albumin depletion that minimally affects the remaining plasma proteins. There are several strategies for achieving this, viz: chromatography [37][38][39][40][41][42], ammonium sulfate precipitation [43,44], ethanol precipitation, and changes in pH [45] to exemplify a few.…”